Endomicroscopy refers to the use if highly miniaturised point scanning confocal microscopes to examine the microscopic structures of tissue in vivo: (endo: introduced into the body, microscopy: visualisation of the tissue structures at the sub-cellular level). The miniaturised confocal microscopes are small enough to be introduced into the body either through natural orifices or using minimally invasive techniques whereby the endomicroscope probe is introduced via a very small incision. Endomicroscopy enables in vivo histology without taking biopsies or sacrificing resolution or image quality.

The FIVE 1 is highly miniaturised fluorescence in vivo confocal endomicroscopy system developed by Optiscan for use in pre-clinical animal research. This system employs the same technology as used in Optiscan’s highly successful, and world leading clinical systems. The FIVE 1 offers the same ultra-high resolution in vivo confocal microscopy capabilities as the clinical systems, albeit in a package developed specifically for animal research. The FIVE 1 system offers the highest resolution in vivo imaging capabilities available.

The FIVE 1 is a not an approved medical device, and has been specifically designed for in vivo examination imaging of animals used in pre-clinical research, and ex vivo tissues. Optiscan has developed other products for a number of clinical applications, which have been approved for clinical use with certain indications. Optiscan has an active clinical research program, and continues to develop additional clinical systems for use in new clinical application areas. If you are involved in translational research and you want to use the same imaging technology in both animal and human studies, use the FIVE 1 system for animal studies and contact Optiscan (clinical@optiscan.com) for information regarding availability of medical devices using the same technology.

The FIVE 1 is a full point scanning confocal microscope that has been highly miniaturised without sacrificing the performance or the optical sectioning capabilities that allow the imaging plane to be dynamically positioned anywhere between the surface of the tissue and approximately 250um below the surface. The FIVE 1 provides sub-organelle level resolution over a very large field of view (compared to the FOV size normally associated with this resolution on a regular bench-top confocal system)

The FIVE 1 is a full point scanning confocal microscope that has been highly miniaturised without sacrificing the performance or the optical sectioning capabilities that allow the imaging plane to be dynamically positioned anywhere between the surface of the tissue and approximately 250um below the surface. The FIVE 1 provides sub-organelle level resolution over a very large field of view (compared to the FOV size normally associated with this resolution on a regular bench-top confocal system)

Yes. Since its release in November 2006, FIVE 1 specific studies continue to be regularly published in peer-reviewed scientific publications, including preeminent publications which have been featured on the cover of major journals such as Gastroenterology. Please refer to the About section of the website for a list of these publications.

The FIVE 1 is Optiscan’s latest pre-clinical animal research instrument in Optiscan’s 20+ year history of miniaturised fibre optic confocal technology. Over this period, there is an extensive publication track record of in vivo confocal microscopy applications using Optiscan instruments across a wide range of applications. As the FIVE 1 is the latest generation technology, it’s performance exceeds that of former instruments, and can be applied to all of the in vivo applications previously published using its predecessors.

The resolution of the FIVE 1 is second to none: The FIVE 1 system offers the highest resolution in vivo imaging capability available.

The FIVE 1 system has a lateral resolution of better than 0.7µm, and an axial resolution (optical slice thickness) of 7.0µm (compared with a typical histological section of 10-15µm used in conventional histology). The field of view is 475µm x 475µm.

The imaging depth is dynamically variable, from the surface of the tissue to approximately 250µm below the surface in 4µm increments. It is not necessary to change endomicroscope probes to image at different depths. The operator can dynamically adjust imaging depth to interrogate the tissue in 3 dimensions.

The resolution of the FIVE 1 is second to none: The FIVE 1 system offers the highest resolution in vivo imaging capability.

The FIVE 1 system has a lateral resolution of better than 0.7µm, and an axial resolution (optical slice thickness) of 7.0µm (compared with a typical histological section of 10-15µm used in conventional histology). The field of view is 475µm x 475µm.

The resolution of the FIVE 1 is second to none: The FIVE 1 system offers the highest resolution in vivo imaging capability. Bacteria have been observed in the gastrointestinal tract, and have been reported in published studies using the FIVE 1 system.

The FIVE 1 system has a lateral resolution of better than 0.7µm, and an axial resolution (optical slice thickness) of 7.0µm (compared a typical histological section of 10-15µm used in conventional histology). The field of view is 475µm x 475µm.

The imaging depth is dynamically variable, and it is possible to image from the surface of the tissue to approximately 250um below the surface in 4um increments using the same endomicroscope probe; ie.it is not necessary to change endomicroscope probes to image at different depths. The operator can dynamically adjust imaging depth to interrogate the tissue in 3 dimensions.

The maximum imaging depth depends upon a number of variables including the type and nature of fluorescent labelling used, the optical properties of the tissue(s) in question, and the emission wavelengths for the fluorophore used.

It is not necessary to change endomicroscope probes to image at different depths. The imaging depth is dynamically variable, and it is possible to image from the surface of the tissue to approximately 250µm below the surface in 4µm increments using the same endomicroscope probe.

Sub-resolvable objects (smaller than the FIVE 1’s resolution) will appear at the size of the lateral resolution limit, ie. they will appear to be 0.7µm even if they are smaller. Objects that are 0.7µm or larger will appear their true size.

The resolution of the FIVE 1 is second to none: The FIVE 1 system offers the highest resolution in vivo imaging capability.

The FIVE 1 system has a lateral resolution of better than 0.7µm, and an axial resolution (optical slice thickness) of 7.0µm (compared with a typical histological section of 10-15µm used in conventional histology). The field of view is 475µm x 475µm.

The imaging depth is dynamically variable, from the surface of the tissue to approximately 250µm below the surface in 4µm increments. It is not necessary to change endomicroscope probes to image at different depths. The operator can dynamically adjust imaging depth to interrogate the tissue in 3 dimensions.

The imaging depth is dynamically variable, and it is possible to image from the surface of the tissue to approximately 250µm below the surface in 4µm increments using the same endomicroscope probe. It is not necessary to change endomicroscope probes to image at different depths.

The FIVE 1 is a single channel system, with 488nm illumination, and a single PMT detector (with 2 user selectable detection bands: either 505–750nm or 550–750nm). This enables extremely simple “point & shoot” operation, with fully automated self calibration. While the FIVE 1 is a single channel instrument, it is still possible to perform a wide range of multi-label experiments (see “Is it possible to perform multi-label experiments (the use of two or more different fluorophores targeting specific kinds of cells) using the FIVE 1 system?”)

FIVE 1 uses a single wavelength excitation at 488 nm and detects the fluorescent emission across a broad bandwidth onto a single detector. Therefore when >1 fluorophore is used to label tissues under investigation, the FIVE 1 will simultaneously detect all fluorophores onto the same detector but they will not be spectrally separated. However, where multiple fluorophores are used to simultaneously label different structures within the tissue, it is possible to distinguish 2 or 3 different labels from one another based on morphology. For example if the fluorophores each label:

• Different cell types with distinct morphology (eg tumour cells vs vasculature or nerves vs blood vessels or vascular endothelium & smooth muscle cells vs labelled peripheral blood mononuclear cells vs RBC cell tracking).
• Different cellular components (eg nuclei vs membranes),
• Different cellular functions (eg labelling of the plasma within blood vessels to contrast the microvasculature & unlabelled red blood cells vs tracking of specifically labelled leukocytes or introduced cells such as stem cells or GFP expressing cultured cells)

It is very common to perform multi-label experiments using the FIVE 1, and numerous studies have been published using this technique to answer complex biological questions. Applications include:

• Cell tracking studies, particularly where populations of cells have been very specifically labelled ex vivo/in vitro and introduced into an animal (eg tracking of cultured/transgenic cells)
• Study of cell –cell interactions
• Cell – tissue interactions eg cell adhesion & aggregation in the microvasculature
• Inflammatory responses
• Acute tissue injury in the airways
• Angiogenesis & relationship to surrounding tumour cells
• Localising pancreatic β islet cells relative to pancreatic microvasculature
• Thrombosis studies
• Microvascular architectural studies etc.

Co-localisation studies where the fluorophores will label the same structure (eg labelling 2 different proteins on the same organelle) are not possible as the FIVE 1 does not spectrally differentiate one fluorophore from another and therefore it is not possible to distinguish whether the labelled structures are labelled with one, ore more than one label.

The FIVE 1 is a single channel system, with 488nm illumination, and single PMT detector (with 2 user selectable detection bands: either 505–750nm or 550–750nm). Therefore when >1 fluorophore is used to label tissues under investigation, the FIVE 1 will simultaneously detect all fluorophores onto the same detector but they will not be spectrally separated. It is possible to apply pseudo colour to any acquired image.

488nm illumination maximises the utility of the FIVE 1 system, as it allows access to the widest range of applications (ie maximum number of fluorescent contrast agents available), offers higher resolution imaging and enables sub surface imaging to approximately 250µm or so below the surface of the tissue. While longer wavelength illumination (eg NIR: Near Infra Red) does allow slightly deeper imaging depths to be reached, the resolution decreases sharply with longer wavelengths, and as there are far fewer fluorescent labels available which are excited in the red & NIR regions, the range of application choices is limited.

The FIVE 1 has been highly optimised to image the widest range of fluorophores available, which corresponds to blue illumination (488nm) and a very wide detection bandwidth (505 – 750nm). While alternate wavelengths for either the laser or detection system are not currently offered, we do invite you to contact Optiscan to discuss your requirements.

FIVE 1 is quick and easy to set up, and no calibrations are required. The system has been designed to be set up on a trolley if required to enable the system to be moved from lab to lab, or within a busy operating suite with ease. Of course the FIVE 1 is equally at home set up on a bench.

The FIVE 1 system is “plug & play”, and no calibrations are required. The system self-calibrates automatically without any input from the operator.

The FIVE 1 is a full point scanning confocal microscope that has been highly miniaturised without compromising performance (ie no compromises in resolution or variable imaging depth capabilities). This is achieved using Optiscan’s patented fibre optic technology: please refer to “how it works”.

Fibre bundle probes employ a bundle of several thousand optical fibres. Laser light is scanned across the proximal end of the fibre bundle, which results in the laser light only travelling through the core of only 1 fibre at any instant in time. When the distal tip of the bundle is placed in contact with the tissue, each individual fibre makes a discrete fluorescence intensity measurement, and by collecting the signal from each fibre in the bundle, an image is formed. While fibre bundles allow very small diameter probes with no moving parts at the distal end, they sacrifice resolution and do not allow variable imaging depth: an essential feature of confocal microscopy. Resolution of fibre bundles is limited due to the small number of fibres in the bundle (compare to the analogue scanning of a point scanning confocal system), and the spacing between the cores of the fibre. For a detailed explanation of fibre optic confocal microscopy and the use of single fibre point scanning technology and fibre bundle technology, refer to:

Delaney & Harris. Fiber-Optics in Scanning Optical Microscopy. In Handbook of biological confocal microscopy, 3rd Edition, Ed J.B. Pawley. 2006, Springer. pp501-515.

Currently the endomicroscope probes for the FIVE 1 system come in a range of different form factors for different applications, but they are currently all the same diameter. Optiscan has an active development program which includes further miniaturisation without sacrificing performance or the variable imaging depth capabilities that are so crucial for in vivo endomicroscopy. The diameter of the FIVE 1 probes is primarily determined by the lens system used in the probes, and it is the lens which provides the unrivalled performance of the FIVE 1 system, ie no compromises in either resolution or the dynamically variable imaging depth capabilities of confocal microscopy.

While it is possible to make much smaller diameter probes using fibre bundle technology, this approach comes with significant compromises in resolution, and prohibits subsurface imaging: the smallest fibre bundle probes only allow imaging at the tissue surface. To enable sub-surface imaging using a fibre bundle probe, a lens must be incorporated onto the distal tip of the bundle which increases the diameter but still does not allow variable depth control.

The standard ultra-high resolution FIVE 1 endomicroscope probes are routinely used for imaging structures at or near the surface of the brain of small animals (and also large animals). As the maximum imaging depth of confocal microscopy is limited to approximately 250µm below the surface, it is necessary to surgically access the brain.

In some experiments with an exploratory workflow, it can be highly desirable to have the freedom of movement afforded by using the endomicroscopes hand-held.

Where it is desirable to observe the same microscopic tissue structures over long periods (eg continuous observation of blood flow or cell tracking in a capillary bed), some operators prefer to use some form of manipulator. The cylindrical form factor of the FIVE 1 endomicroscope probes enables them to be held using a variety of mechanical aids which can be as simple as a retort stand and clamp, to a micromanipulator with multi-axis micrometer positioning, or fitted to the manipulators on a stereotactic frame for precise positioning in brain imaging.

The term magnification originates from traditional microscopy where objects were magnified using a system of lenses and viewed with an eyepiece. With digital imaging, the magnification from the object to the displayed image depends on the size that the image is displayed (eg on the size of the monitor, electronic zoom etc).

Therefore the parameter that is referred to is the Field Of View (FOV), as FOV does not change when the image is displayed on monitors of different sizes. The FOV refers to the size of the area that is represented in the image, or in other words the area of tissue that is actually scanned to collect the image. In the case of the FIVE 1 the FOV is 475µm x 475µm.

As the FIVE 1 system allows the operator to dynamically adjust imaging depth in 4µm increments, it is possible to collect a series of images that span a depth range (3D stack or Z series), and this can be used for 3D reconstruction if desired. The suitability of in vivo data for 3D reconstruction and 3D analysis depends on the structures that have been labelled within the tissue of interest, and how much tissue movement is present.

While it is possible to collect Z series, more typically the FIVE 1 workflow is more like “ultrasound”, where the operator interacts with the tissue, and interrogates the tissue in 3 dimensions by observing not only the specific structures of interest, but also those surrounding them (eg deeper/superficial, left/right etc) to obtain a thorough spatial understanding of the microscopic architecture and cellular relationships.

For all measurement systems, including in vivo microscopy, there is a trade off between spatial resolution & temporal resolution (ie acquisition of high resolution images takes time, and by reducing the amount of data collected enables increased speed / frame rate).

The FIVE 1 system acquires approx 840 lines/second, with 1024 measurements/line (total of approx 860,000 measurements/sec). For fluorescence imaging, speed is limited by the rate at which photons leave the tissue, and this dictates the number of measurements/sec that can be captured, or in other words the number of pixels/sec.

Due to the ultra high resolution of the FIVE 1 optics, and the fact that the FIVE 1 is a point scanning confocal microscope, the FIVE 1 has the performance to produce megapixel images that are worthy of digital zoom (1024 x 1024 or 1024 x512 pixels), resulting in frame rates of 0.8 & 1.4 images/sec respectively.

On the other hand, technologies with much lower optical resolution such as fibre bundle probes cannot provide sufficient resolution for megapixel sampling, and therefore often offer higher frame rates.

Two frame rates are available for FIVE 1. Frame rate is varied by changing the sampling resolution, ie. the number of lines collected per image. The FIVE 1 offers 1024 x 1024 or 1024 x 512 pixel images, resulting in frame rates of 0.8 & 1.4 images/sec respectively.

FIVE 1 measures fluorescence intensity. The fluorescence intensity is proportional to the local concentration of the fluorophore, unless saturation of the fluorophore has been reached.

FIVE 1 is a fluorescence confocal microscope which excites fluorescent molecules using 488nm laser light, and detects the resultant emission in the visible part of the spectrum (505-750nm). While there are some naturally occurring fluorescent substances present within many animal tissues, most are in very low concentrations, and are most often UV excited, therefore not excited / detected by the FIVE 1. Therefore it is normal practice to use an exogenously applied fluorescent contrast agent(s) to label tissues/cells/organelles of interest. Other techniques that can also be used include imaging of GFP or YFP expression in transgenic animals or cultured transgenic cells that have been administered to an animal.

While there are some naturally occurring fluorescent substances present within many animal tissues, most are in very low concentrations, and are most often UV excited, therefore not excited / detected by the FIVE 1. Therefore it is normal practice to use an exogenously applied fluorescent contrast agent(s) to label tissues/cells/organelles of interest.

No.FIVE 1 is a fluorescence confocal microscope which excites fluorescent molecules using 488nm laser light, and detects the resultant emission in the visible part of the spectrum (505-750nm).

Autofluorescence normally does not interfere with specific labelling of cells using exogenous fluorescent contrast agents. While there are some naturally occurring fluorescent substances present within many animal tissues, most are in very low concentrations, and are most often UV excited, therefore not excited / detected by the FIVE 1. As a result, typically there is very little background fluorescence from mammalian tissue.

Two frame rates are available for FIVE 1. Frame rate is varied by changing the sampling resolution, ie. the number of lines collected per image. The FIVE 1 offers 1024 x 1024 or 1024 x 512 pixel images, resulting in frame rates of 0.8 & 1.4 images/sec respectively.

Basically, the FIVE 1 can image any blue (488nm) excited fluorophore that emits in the visible or far red part of the spectrum. There is a wide range of commercially available fluorophores that can be used to label cells of interest in vivo. The following is a brief list of fluorophores that have been used with the FIVE 1:

Fluorescent dyes

Syto 13

Carboxyfluorescein

CFSE FITC-Albumin

FITC-Dextrans

Methylene green

AlexaFluor 488

Acrydine orange

Acriflavine

Proflavine

Euflavine

Lucifer Yellow

In vivo immunolabelling using FITC-labelled antibodies

FITC-labelled oligonucleotides

Carboxyfluorescein labelled receptor ligands

Tetracylines

Cresyl violet

Fluorescently labelled lectins

Fluorescent protein expression either in transgenic animals, or by administering cultured cells expressing fluorescent proteins such as

• GFP
• eGFP
• YFP

Many of the fluorescence labelling techniques used “on the bench” can be applied to in vivo experiments, using many of the 1000s of blue excited visible emitting fluorophores that are commercially available. In most cases that protocols are very simple and straightforward as the living tissue often facilitates the labelling process.

Please refer to the bibliography and applications notes for detailed examples of in vivo labelling protocols used in a range of different applications.

Our application specialists have many years of experience with in vivo endomicroscopy in a wide range of applications and can provide advice.

Many of the fluorescence labelling techniques used “on the bench” can be applied to in vivo experiments, using many of the 1000s of blue excited visible emitting fluorophores that are commercially available. In most cases that protocols are very simple and straightforward as the living tissue often facilitates the labelling process.

Please refer to the bibliography and applications notes for detailed examples of in vivo labelling protocols used in a range of different applications.

Our application specialists have many years of experience with in vivo endomicroscopy in a wide range of applications and can provide advice.

The most basic image comprises quantification of the morphology and fluorescence intensity as the signal is directly proportional to the amount of light detected. Like all digital images, quantitative image analysis is possible to determine a range of image parameters such as cell (object) counting, length & area measurement, integrated intensity measurements, shape analysis etc. The FIVE 1 system is supplied with image analysis tools, and also provides a range of image file formats to enable image analysis using your favourite 3rd party package.

The FIVE 1 system is provides a range of standard image file formats to enable image transfer to your favourite 3rd party image analysis software.

The FIVE 1 is PC based. It is not available for the Mac.

The FIVE 1 is a fluorescence only confocal endomicroscopy system, and does not provide reflectance (backscatter) imaging.

The maximum depth for confocal microscopy is limited to approximately 250µm below the surface. Therefore the structures of interest must be located within 250µm of the contact surface. As the skin in most animals (even small animals such as mice) is several hundred microns thick, it is not possible to image internal organs through the skin. When the endomicroscope probe is placed in contact with the skin, it is possible to image the microscopic structure of the skin itself. The endomicroscope must be placed in gentle contact with the tissue; if not, there will be no image. Therefore it is necessary to access the tissue of interest surgically if it cannot be accessed via a natural orifice. This can be done using minimally invasive techniques whereby the endomicroscope probe is passed through a tiny incision, and the tissue of interest is imaged laparoscopically, or alternatively a larger incision can be made to allow greater access and easy visualisation of the imaging field.

Obviously where the tissue movement is minimal, obtaining motion artefact-free images is easier. However, as the tip of the endomicroscope probe must be placed in gentle contact with the tissue, the endomicroscope probe and tissue can move together and thus the relative movement between the tissue and the endomicroscope probe is significantly reduced, and in most cases eliminated. Therefore, it is possible to image dynamic tissues with relative ease even though they are moving continuously.

The maximum depth for confocal microscopy is limited to approximately 250µm below the surface. Therefore the structures of interest must be located within 250µm of the contact surface. Where internal tissues (eg peritoneal organs) are to be imaged, it is necessary to access the tissue of interest surgically if it cannot be accessed via a natural orifice. This can be done using minimally invasive techniques whereby the endomicroscope probe is passed through a tiny incision, and the tissue of interest is imaged laparoscopically, or alternatively a larger incision can be made to allow greater access and easy visualisation of the imaging field.

Where internal tissues (eg peritoneal organs) are to be imaged, it is necessary to access the tissue of interest surgically if it cannot be accessed via a natural orifice. This can be done using minimally invasive techniques whereby the endomicroscope probe is passed through a tiny incision, and the tissue of interest is imaged laparoscopically, or alternatively a larger incision can be made to allow greater access and easy visualisation of the imaging field. The maximum depth for confocal microscopy is limited to approximately 250µm below the surface. Therefore the structures of interest must be located within 250µm of the contact surface.

Generally, no attempt is made to prevent tissue movement, but rather the tissue and probe are allowed to move together.

The endomicroscope must be placed in gentle contact with the tissue; if not, there will be no image. When the endomicroscope probe is in contact with the tissue, the tissue and probe can move together, and thus the relative movement between the tissue and the endomicroscope probe is significantly reduced, and in most cases eliminated. Therefore, it is possible to image dynamic tissues with relative ease even though they are moving continuously.

Many of the fluorescence labelling techniques used “on the bench” can be applied to in vivo experiments, using many of the 1000s of blue excited visible emitting fluorophores that are commercially available. In most cases that protocols are very simple and straightforward as the living tissue often facilitates the labelling process.

Please refer to the bibliography and applications notes for detailed examples of in vivo labelling protocols used in a range of different applications.

Our application specialists have many years of experience with in vivo endomicroscopy in a wide range of applications and can provide advice.

Simple contact between the tip of the endomicroscope probe and the tissue minimises relative movement between the tissue and endomicroscope, thus enabling stable microscopic images to be obtained from living tissue in vivo even though the tissue is moving.

While the tip of the endomicroscope probe must touch the tissue to enable imaging, the tip of the endomicroscope probes has been designed with a sufficiently large surface area, and gently curing profile to minimise tissue damage and perforation risk.

The endomicroscope only needs to touch the tissue lightly when imaging. It is not necessary to apply pressure, and varying pressure does not affect imaging depth.

The imaging depth is varied by the operator via a foot control that moves the optics within the distal tip of the FIVE 1 endomicroscope probe to change imaging depth.

In most soft tissues, the endomicroscope probe does not need to be perpendicular to the surface of the tissue, and in most cases can be placed at angles as low as 30 degrees to the tissue surface. It is also possible to image the luminal wall of hollow (tubular organs) as the endomicroscope probe is passed through the lumen.

In some firm tissues such as cartilage, the endomicroscope probe must be oriented perpendicular to the tissue surface to provide an adequate interface with the tissue.

Currently, all of the FIVE 1 endomicroscope probes are “end viewing”. It is possible to image the luminal wall of hollow (tubular organs) as the endomicroscope probe is passed through the lumen, as most soft tissues are distensible and allow an adequate tissue interface even with an end viewing probe.

The FIVE 1 endomicroscope probes are immersible for cleaning and (chemical) disinfection. This allows the probes to be cleaned with a range of common chemicals used for disinfection/sterilisation.

The FIVE 1 endomicroscope probes are compatible with a range of common chemicals used for disinfection/sterilisation. The probes are immersible.

The FIVE 1 is not a medical device, so it has not been used in clinical trials. However, the FIVE 1 uses the same technology as Optiscan’s other clinical instruments, and therefore has the same performance as the clinical systems. For more information on Optiscan’s clinical trial activities and clinical instruments, please contact clinical@optiscan.com

The FIVE 1 is supplied with dedicated control software used to operate the instrument, acquire data and store the procedure results in an integral database for easy retrieval and review. Image analysis and manipulation tools are also provided for easy quantification of data.

FIVE 1 is designed for in vivo imaging in a wide range of animals used in research, including the larger animal models such as dogs, rabbits, sheep, pigs etc. In fact, longer laparoscope style probes are available for minimally invasive applications in larger animal models.

FIVE 1 is designed for in vivo imaging in a wide range of laboratory animals, including mice. Many studies are performed in mice across a wide range of applications, and such studies are routinely published.

Optiscan’s endomicroscopy technology that is used in the FIVE 1 is the highest resolution in vivo imaging modality. The FIVE 1 offers sub-cellular resolution in vivo that is second to none. Not only is it possible to clearly image sub-cellular structure, it is possible to image sub-organelle structures in vivo at, and below the tissue surface.

The FIVE 1 is used across a very wide range of applications ranging from basic morphology assessment and acute injury experiments to longitudinal studies in tumour progression, biomaterials compatibility research, investigation of disease models, cell tracking etc. These are performed in a range of animal models from mice to larger animals such as rabbits, dogs, cows, sheep and pigs. The FIVE 1 is also used to examine tissues ex vivo. Please refer to the bibliography for the latest list of peer reviewed publications.

Confocal microscopy is limited to approximately 250m below the tissue surface. The imaging depth is dynamically variable, and the operator can vary the imaging depth between the tissue surface to approximately 250m below the tissue surface. However, the actual depth that can be achieved in a specific experiment will depend strongly upon the light scattering and absorption properties of the tissue.

The FIVE 1 is designed to perform in vivo histology without taking biopsies. This provides sub-cellular level detail of tissues & cells in living animals. Due to the very high magnification and resolution capabilities of the FIVE 1, and the fact that on the microscopic level there is tissue movement, the FIVE 1 captures an image with a single scan across the tissue (<1sec/image). The whole body fluorescence imaging systems are designed to detect very low level fluorescence signals from within a whole mouse. Due to the very low level fluorescence detected by these systems, they integrate the fluorescence signal both spatially (ie low resolution) and over very long exposure times (minutes) to build up an image. These are used to localise fluorescent signals to the organ level.

A range of endomicroscope probes are available for use with the FIVE 1, and these have been designed with different form factors that suit particular types of experiments. For example laparoscope style probes are available for minimally invasive experiments in large animals, small animal probes and pen style probes that can be applied to a very wide range of applications.

The operator is able to dynamically adjust imaging depth in 4m increments from the surface of the tissue to approx 250m below the surface, however due to refractive index changes within individual tissues which can perturb accurate assessment of imaging depth, the absolute imaging depth is not reported to the operator. It is possible to estimate the imaging depth.

The processes used to prepare histological slides such as fixation, dehydration, staining, rehydration and mounting cause distortion of the specimen (eg shrinkage and cracking). In vivo endomicroscopy looks at tissue in its natural, living state, and hence the cells sometimes appear different to what is observed in conventional histology.

For conventional histology, tissue sections are typically cut to produce transverse sections. Endomicroscopy produces en face images (parallel to the surface), and therefore some cells/tissue structures simply look different due to the orientation of the image plane relative to the 3D structure of the tissue.

These are two entirely different technologies that have different modes of operation, and hence produce different results. A confocal microscope is designed to “throw away” unwanted signal originating from “out-of-focus” regions, whereas a regular fluorescence microscope detects all signal collected by the lens (even if it originates from outside the focal plane) and integrates the signal.

A regular fluorescence microscope uses a broad spectrum illumination which excites fluorophores in the specimen across a wider spectrum than a monochromatic laser source. On top of this, a regular fluorescence microscope does not optically section, rather it integrates the fluorescence signal from throughout the entire thickness of the specimen. Overall this equates to a broad general excitation, and detection from a large volume of tissue.

Confocal microscopy on the other hand uses monochromatic illumination to excite over a discrete wavelength range of just a few nm, and only detects the resultant fluorescence from a discrete focal volume of a few femtolitres at a time within the tissue (ie the fluorescent signal is detected from a much smaller volume within the tissue.